Nucleic acids are synthesized using solid supports and 5′-dimethoxytrityl protected phosphoramidite reagents (Beaucage, 1981; Caruthers, 1983). After completion of a synthesis, they are cleaved from the synthesis supports either as their 5′-unprotected form (DMT-off oligonucleotides) or as their 5′-dimethoxytrityl protected form (DMT-on oligonucleotides).
DMT-off oligonucleotides, notably long oligonucleotides, are desalted or purified by way of high-performance liquid chromatography (Ikuta, 1984) or polyacrylamide gel electrophoresis. DMT-on oligonucleotides are best purified by Solid-Phase Extraction (SPE) using disposable cartridges prepacked with reversed-phase sorbents such as silane-modified controlled pore glass (CPG) or cross-linked polystyrenes (Andrus, 1993; Semenyuk, 2006). The said sorbents reversibly adsorb DMT-on oligonucleotides by taking advantage of the hydrophobic handles provided by the 5′-dimethoxytrityl groups (Cashion, 1973) while polar DMT-off failures, salts and organic impurities are effectively eliminated in the flow-through and subsequent washing steps. Subsequent on-cartridge detritylation of DMT-on oligonucleotides and elution yield the corresponding purified DMT-off oligonucleotides.
SPE purification is the method of choice for full length DMT-on oligonucleotides with fragment less than 40 bases. Its shortcoming stems from the plurality of DMT-on species generated during synthesis and deprotection. Full length oligonucleotides are invariably contaminated with short length DMT-on oligonucleotides arising from depurination (Horn, 1988) or branching (Pon, 1985). Increased contaminations in long oligonucleotides hinder the use of SPE columns. Commercial suppliers recommend long oligonucleotides to be purified by HPLC or PAGE, therefore preventing their widespread use due to high processing costs.
Methods and reagents capable of isolating long oligonucleotides from short length DMT-on oligonucleotides, in a manner that avoids limitations to the current systems, are urgently needed. The present invention describes a multimodal cartridge system or Multi Layer Chromatography (MLC) that efficiently separates full length DMT-on nucleic acids from hydrophobic contaminants. The said MLC system is readily amenable to a high throughput purification of long oligonucleotides up to 200-mers and could substantially contributes to the field of molecular biology (gene synthesis, hybridization probes, long primers etc) by making pure long oligonucleotides quickly available at a fraction of the current costs.